Half of all neurons produced during embryogenesis undergo apoptotic death shortly before birth or soon thereafter. Neuronal death with characteristics similar to those seen during development also occurs after stroke and in neurodegenerative diseases such as Alzheimer's disease. Similarities between developmental death and death caused by insult or disease suggests comparable processes kill neurons in both situations and that information gained about mechanisms of developmental death may aid in understanding and treating pathological death. We use two cell culture models to investigate mechanisms of apoptosis during neuronal development: 1) sympathetic neurons deprived of nerve growth factor and, 2) cerebellar granule neurons deprived of serum in low potassium medium. Data generated during the initial funding period of this grant shows that there is a dramatic increase in mitochondrial-derived reactive oxygen species (ROS) during the apoptotic death of both of these cell types. These ROS lie downstream of the pro-apoptotic protein, Bax, which induces apoptosis by causing release of apoptogenic factors from the mitochondria into the cytoplasm. Our evidence suggests that the ROS are critical for this release. The goal of this research proposal is to understand the role of these ROS in neuronal apoptosis. The first four specific aims will be done in sympathetic neurons. Specific aim 1 will test the hypotheses that Bax induces elevated ROS by increasing production of superoxide by mitochondria. The goal of specific aim 2 is to test the hypothesis that caspase proteases increase ROS by attacking mitochondrial respiratory complexes. Specific aim 3 will test the hypothesis that the Bax-induced ROS lie downstream from the MLK/JNK kinase pathway. Experiments in specific aim 4 will test the hypothesis that the ROS cause release of apoptogenic factors from mitochondria by opening the outer mitochondrial membrane channel, VDAC. Specific aim 5 will test the generality of the first four specific aims in the cerebellar granule system. We shall use genetic, biochemical, and confocal microscopic techniques to investigate these hypotheses. These studies will provide clear answers about the mechanism by which Bax causes increased ROS during neuronal apoptosis and how these ROS contribute to cell death. They will further our long-term goals of understanding mechanisms of apoptosis and of identifying ways of manipulating this death.